Circulating gut microbial metabolites and risk of coronary heart disease: A prospective multi-stage metabolomics study.

The discovery stage identified 73 circulating microbiota-related metabolites associated with incident CHD at a false discovery rate less than 0.10.

• Discovery involved untargeted plasma metabolite profiling of 896 incident cases and 896 age-/sex-/race-matched controls (~300 pairs per race: Black, White, Asian).
• Cohorts included the Southern Community Cohort Study (SCCS) and the Shanghai Women's Health Study and Shanghai Men's Health Study (SWHS/SMHS).
• Conditional logistic regression estimated odds ratios of incident CHD per standard deviation metabolite increase.
• Covariates adjusted included age, sex, race, education, income, smoking status, alcohol consumption, physical activity, diet quality, and BMI.

Of 61 metabolites available for in-silico validation, 24 showed a significant association in the same direction as the discovery stage.

• In-silico validation was conducted in ARIC (N=3,539; 663 cases; baseline: 1987-1989) and MESA (N=3,860; 446 cases; baseline: 2000-2002).
• Significance threshold for validation was p<0.05 in the same direction as discovery.
• Cox regression was used in ARIC and MESA with a cohort design.
• Not all 73 discovery metabolites were available for validation due to differences in metabolomic assay coverage across stages.

Five metabolites quantified by targeted assay were significantly associated with incident CHD with ORs per SD ranging from 1.18 to 1.27: imidazole propionate, 3-hydroxy-2-ethylpropionate, 4-hydroxyphenylacetate, trans-4-hydroxyproline, and 3-hydroxybutyrate.

• The quantitative stage applied a targeted assay to a new set of 864 cases and 864 age-/sex-/race-matched controls (~260-340 pairs per race) from the SCCS and SWHS/SMHS.
• OR per SD ranged from 1.18 to 1.27 after adjustment for sociodemographics, lifestyles, and BMI.
• The targeted assay quantified eight of the 24 validated metabolites, of which five were significantly associated.
• Associations were assessed using conditional logistic regression in a nested case-control design.

Four additional gut microbial metabolites measured by the targeted assay were significantly associated with incident CHD: trimethylamine N-oxide (TMAO), phenylacetyl-L-glutamine, 4-hydroxyhippuric acid, and indolepropionate.

• These four metabolites were among eight other promising microbial metabolites measured by the targeted assay beyond the primary 24 validated metabolites.
• These metabolites were part of the broader targeted quantitative assay stage applied to 864 cases and 864 matched controls.
• These findings were reported alongside the five primary validated metabolites as significant associations.

Most associations between microbial metabolites and CHD were consistent across participant subgroups defined by demographics, lifestyles, metabolic disease history, family CHD history, and follow-up time.

• Subgroup analyses examined consistency by demographics, lifestyles, metabolic disease history, family CHD history, and follow-up time.
• Some potential effect modifications were found by race, age, obesity status, and follow-up time.
• The study enrolled participants from Black, White, and Asian racial groups (~300 or ~260-340 pairs per race across stages).
• The mean time between enrollment and CHD diagnosis was 5.6 (3.8) years in SCCS, 6.9 (4.4) years in SWHS/SMHS, 15.0 (7.4) years in ARIC, and 8.0 (4.9) years in MESA.

The study used a multi-stage design spanning five prospective cohorts across diverse racial and geographic populations to systematically examine associations between circulating microbial metabolites and incident CHD.

• Five cohorts were used: SCCS, SWHS, SMHS, ARIC, and MESA.
• The study included Black, White, and Asian participants.
• Baseline enrollment periods ranged from 1987-1989 (ARIC) to 2002-2009 (SCCS).
• Three stages were employed: untargeted metabolomics discovery, in-silico validation, and targeted quantitative assay validation.